Persoonia 39, 2017: 74–90 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE https://doi.org/10.3767/persoonia.2017.39.04 Phylogeny of the order Phyllachorales (Ascomycota, Sordariomycetes): among and within order relationships based on five molecular loci M. Mardones1,2, T. Trampe-Jaschik1, S. Oster1, M. Elliott3, H. Urbina4, I. Schmitt1,5, M. Piepenbring1 Key words Abstract The order Phyllachorales (Pezizomycotina, Ascomycota) is a group of biotrophic, obligate plant parasitic fungi with a tropical distribution and high host specificity. Traditionally two families are recognised within this order: ancestral state reconstruction Phyllachoraceae and Phaeochoraceae, based mostly on morphological and host characteristics. Currently, the plant parasitic position of the order within the class Sordariomycetes is inconclusive, as well as the monophyly of the order, and tar spot fungi its internal phylogenetic structure. Here we present a phylogeny of the order Phyllachorales based on sequence Telimenaceae data of 29 species with a broad host range resulting from a wide geographical sampling. We inferred Maximum Likelihood and Bayesian phylogenies from data of five DNA regions: nrLSU rDNA, nrSSU rDNA, ITS rDNA, and the protein coding genes RPB2, and TEF1. We found that the order Phyllachorales is monophyletic and related to members of the subclass Sordariomycetidae within Sordariomycetes. Within the order, members of the family Phaeochoraceae form a monophyletic group, and the family Phyllachoraceae is split into two lineages. Maximum Likelihood ancestral state reconstructions indicate that the ancestor of Phyllachorales had a monocotyledonous host plant, immersed perithecia, and a black stroma. Alternative states of these characters evolved multiple times independently within the order. Based on our results we redefine the family Phyllachoraceae and propose the new family Telimenaceae with Telimena erythrinae as type species, resulting in three families in the order. Species of Telimena spp. occur in several monocotyledonous and eudicotyledonous host plants except Poaceae, and generally have enlarged black pseudostroma around the perithecia, a character not present in species of Phyllachoraceae. Article info Received: 19 September 2016; Accepted: 1 March 2017; Published: 20 June 2017. INTRODUCTION unitunicate asci of cylindrical to clavate shape, with an ascus crown and an inconspicuous apical ring not staining blue in Phyllachorales is an order of biotrophic, obligate plant parasitic iodine; and globose to filiform ascospores, which in most spe- fungi in the class Sordariomycetes, i.e., inoperculate pyreno- cies are hyaline and 1-celled, with only a few genera including mycetes. About 1 226 species are currently accepted in the species with brown or septate ascospores (Parbery 1967, order (Kirk et al. 2008), although 160 000 species have been Cannon 1991, 1997). Although it has been difficult to connect estimated to occur worldwide (Cannon 1997). Phyllachorales asexual and sexual morphs in the order due to their obligately are highly diverse in the tropics, relatively common in disturbed parasitic condition, some species of Phyllachorales have been and natural vegetation, and likewise found in open and forested linked to the asexual genus Linochora (Von Höhnel 1910), which areas (Piepenbring et al. 2011). may be inconspicuous and spermatial in function (Parbery & Species of Phyllachorales are leaf- or steam-inhabiting micro- Langdon 1963, Parbery 1996). fungi with shiny black stromata, which gave them the common Due to their biotrophic nutrition mode, high host specificity is name ‘tropical tar spot fungi’. They are morphologically char- assumed and species concepts are based partly on the identity acterised by: deep black stromata of various shapes (except in and systematic position of the corresponding host plants. Hence species of Polystigma which have brightly coloured stromata); to identify species of Phyllachorales, it is necessary to identify pseudostroma inside the host tissue and usually beneath an epi- the host plant. Traditionally, new species have been described dermal clypeus; perithecia usually strongly melanised that may on the basis of new host records at generic level. However, be superficial, erumpent or immersed in the host tissue (Fig. examination of species of Phyllachorales on the host families 1a–f); thin-walled paraphyses which frequently deliquesce; Poaceae and Fabaceae demonstrated that tropical tar spot spe- cies are not restricted to a single host genus, but may occur on 1 Institute of Ecology, Evolution and Diversity, Faculty of Biosciences, Goethe species belonging to a group of closely related genera (Parbery University Frankfurt am Main, Biologicum, Max-von-Laue-Str. 13, 60439 1978, Cannon 1991, 1997). Species delimitation based mainly Frankfurt am Main, Germany; on host identity may therefore lead to over-splitting of species corresponding author e-mail: [email protected]. (Cannon 1997). 2 Escuela de Biología, Universidad de Costa Rica, San Pedro, 11501 San José, Costa Rica. Phyllachorales are associated with diverse host plants, and 3 Department of Plant Pathology, University of Florida – IFAS, Fort Lauderdale most of the species are linked to angiosperms, with a few ex- Research and Education Center, Davie, FL-33314, USA. ceptions including the lichenicolous Lichenochora species, the 4 Department of Botany and Plant Pathology, Purdue University, West La- fayette, IN-47907, USA. marine algicolous genus Phycomelaina, and some species on 5 Senckenberg Biodiversity and Climate Research Centre (BiK-F), Sencken- ferns and gymnosperms. Within the angiosperms, the following berganlage 25, 60325 Frankfurt am Main, Germany. families are preferentially parasitized: Arecaceae, Fabaceae, © 2017 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. M. Mardones et al.: Phylogeny of Phyllachorales 75 Lauraceae, Melastomataceae, Moraceae, Myrtaceae, and Poa- throughout the history of the group, several authors have ceae (Cannon 1997). Monographs for phyllachoraceous spe- placed phyllachoraceous fungi into various families and orders, cies are available for Arecaceae (Hyde & Cannon 1999), Faba- stressing different morphological and ecological characteristics: ceae (Cannon 1991), and Poaceae (Orton 1944, Parbery Diaporthales (Cannon 1988), Dothideales (Saccardo 1876, 1967). Additional host families studied include Asclepiadaceae Theissen & Sydow 1915), Polystigmatales or Polystigmata- (Pearce et al. 1999), Erythroxylaceae (Cannon & Evans 1999), ceae (Von Arx & Müller 1954, Eriksson 1982, Hawksworth et Proteaceae (Pearce et al. 2001), and Rosaceae (Cannon 1996). al. 1983), Sphaeriales (Nannfeldt 1932, Luttrell 1951, Müller & The genus Phyllachora was introduced on a herbarium label in Von Arx 1962, 1973), and Xylariales (Barr 1983). For a detailed Fuckels exsiccate series ‘Fungi Rhenani’ with a single species, description of the taxonomical history of the order see Cannon P. agrostis (Fuckel 1867 in Cannon 1991), currently accepted (1991) and Pearce & Hyde (2006). as Scirrhia agrostis in Dothideales (Eriksson 1967). Later the The order Phyllachorales comprises the families Phyllachorace- genus Phyllachora was lectotypified with Phyllachora graminis ae and Phaeochoraceae. The family Phyllachoraceae was as generic type (Clements & Shear 1931), and the genus name erected by Theissen & Sydow (1915) and is by far the largest in the sense of Fuckel (1870) was conserved to allow continued family within the order with almost 1 200 described species use in its currently accepted circumscription. The order Phyl- (Kirk et al. 2008). The number of genera varies between 51 lachorales was formally described by Barr (1983). However, (Kirk et al. 2008) and 73 (www.indexfungorum.org). Many of Fig. 1 Perithecia of species of Phyllachorales in different positions in the mesophyll of the leaves. a. Phyllachora graminis (isotype CUP3536) with immersed perithecia and few pseudostroma; b. Coccodiella miconiae (ppMP1342) with superficial perithecia; c. Camarotella costaricensis (MM-21) with erumpent perithecium; d. Polystigma pusillum (MM-113) with brightly coloured stroma; e. Serenomyces phoenicis (F59049) with subcuticular perithecium and without a clypeus; f. Telimena bicincta (epitype MM-133) with immersed perithecia and strongly developed pseudostroma. — Scale bars = 100 µm. 76 Persoonia – Volume 39, 2017 these genera, however, have less than ten species and 27 are Extraction, amplification, and sequencing of DNA monotypic. Phyllachora is the largest genus with 994 species; DNA was isolated directly from hymenia of fresh, recently col- and Coccodiella, Lichenochora, Ophiodothella, Polystigma, lected material or from dry specimens except for